PROJECT SUMMARY The overall objective is to define the cellular mechanisms that contribute to cholestatic liver injury. The ductular reactive (DR) cell population arising in cholestatic liver injury promotes hepatic fibrosis. Our proposal is focused on DR cell apoptosis as a potential therapeutic strategy to attenuate cholestatic liver injury and fibrosis. To this end, we have made several pivotal observations. We observed that tumor necrosis factor- related apoptosis-inducing ligand (TRAIL) receptor (TR), a death receptor, limits the extent of the DR cell population in vivo in a murine model of cholestasis (Mdr2-/- mice) by inducing DR cell apoptosis. This observation indicates that DR cells may be considered ?primed? for apoptosis. As macrophages express TRAIL, we next examined if DR cells by a compensatory counter regulatory process may promote macrophage TRAIL expression thereby limiting their expansion. EpCAM positive reactive cholangiocyte (ERC) organoids from Mdr2-/- mice were developed by us as a DR cell model. ERC organoids release extracellular vesicles (EVs) which contain cargo promoting macrophage TRAIL expression. Specifically, the EVs contain S100A11, a damage-associated molecular pattern (DAMP) protein, which binds scavenger receptors (SR) on macrophages. Accordingly, an inhibitor of the SR termed Mer receptor tyrosine kinase (MerTK) attenuated TRAIL-induction in macrophages by the EVs. Cells primed for TRAIL cytotoxicity are sensitive to BH3 mimetics targeting myeloid cell leukemia 1 (Mcl1), a pro-survival member of the Bcl-2 protein family. Consistently, ERC organoids underwent apoptosis by a BH3 mimetic targeting Mcl1, S63845. Based on these preliminary data, we formulated the CENTRAL HYPOTHESIS that DR cells promote a compensatory counter regulatory process to limit their expansion by inducing macrophage TRAIL expression, and are primed for cell death which can be exploited therapeutically to attenuate cholestatic liver injury. We will now employ current and complementary experimental approaches, and preclinical models to examine this hypothesis. Our SPECIFIC AIMS will test three hypotheses. FIRST, we will test the hypothesis that TRAIL/TRAIL receptor signaling, using conditional knockout mice, directly restrains the DR cell population in cholestasis by mechanisms dependent upon: a) TRAIL receptor expression by DR cells; and b) TRAIL expression by macrophages. SECOND, we will test the hypothesis that ERC organoids release EVs inducing TRAIL expression in macrophages by mechanisms dependent upon: a) the EV cargo S100A11; and b) MerTK activation on macrophages. FINALLY, we will test the hypothesis that ERC organoids and DR cells are primed for cell death such that: a) survival is dependent upon the anti-apoptotic function of Mcl1 in vitro; and b) pharmacologic targeting of Mcl1 in vivo induces DR cell apoptosis thereby attenuating liver injury and fibrosis. This technically and conceptually innovative application, is also significant because it may identify therapeutic strategies for purposefully inducing DR cell apoptosis to limit cholestatic liver and fibrosis.